We will study the roles of PICK1 (Protein Interacting with C Kinase-1) and its regulator, NSF (N-ethylmaleimide Sensitive Factor) in AMPA receptor (AMPAR) trafficking. AMPAR are the major source of excitatory currents in the CNS and changes in AMPAR synaptic abundance regulate synaptic strength. PICK1 and NSF bind to the C-terminal, cytoplasmic domain of the AMPAR GluR2 subunit, and have opposing roles in controlling AMPAR synaptic levels. PICK1 binds to GluR2 via a PDZ domain and colocalizes with AMPAR in endosome-like vesicles in hippocampal pyramidal neurons. PICK1 expression reduces AMPAR synaptic membrane abundance and PICK is implicated in the mechanism of long term depression (LTD), all indicating a role for PICK1 in AMPAR endocytosis. NSF, together with the SNAP proteins, is known for dissociating SNARE complexes. In the case of AMPAR, the interaction of NSF with GluR2 is necessary for maintaining AMPAR currents and synaptic abundance. Our recent work has revealed a novel function for NSF. NSF binds together with the SNAPs to the PICK1-GluR2 complex and displaces PICK1 from GluR2. By displacing an endocytosis factor, NSF maintains AMPAR in the synapse. The Aims of this project are: 1) To determine the structural basis for assembly of the GluR2-PICK1-NSF-SNAP complex and its disassembly by NSF. We will define protein-protein contacts that stabilize the complex and determine whether the SNAPs transmit rotational torque from NSF to PICK1 during disassembly. We will determine the basis for the differential ability of alpha- and beta-SNAPs to disrupt PICK1-GluR2 complexes. 2) To determine the role of PICK1 in AMPAR trafficking. We will identify the starting and destination membranes and the roles of G proteins in the transport mechanism. 3) To determine regulation in vivo by physiologic stimuli, including the role of PICK1 in GluR2/GluR3 constitutive recycling. These studies will reveal basic mechanisms relevant to synapse regulation and memory formation.